Use of somatostatin or one of its analogues for preparing a medicament intended to regulate the ovarian follicular reserve in non-menopausal women

ABSTRACT

The invention primarily relates to the use of somatostatin or one of the agonistic analogs thereof for producing a medicament serving to regulate the ovarian follicular reserve and, in particular, to reduce the depletion of the ovarian follicular reserve over time in non-menopausal women or to the use of an antagonistic analog of somatostatin for producing a medicament serving to accelerate the start of growing of quiescent follicles in non-menopausal women. The invention also relates to in vitro applications of somatostatin and of agonistic and antagonistic analogs thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/575,236, filed Nov. 16, 2006, the disclosure of which is herebyincorporated by reference in its entirety, which claims the benefit ofEuropean Appl. No. EP 03292505, filed Oct. 10, 2003 andPCT/FR2004/002536, filed Oct. 8, 2004, the disclosures of both of whichare hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to the use of somatostatin or one of its agonistanalogues for preparing a medicament intended to regulate the ovarianfollicular reserve in non-menopausal women, or the use of a somatostatinantagonist analogue for preparing a medicament intended to acceleratethe start of growth of the quiescent follicles in non-menopausal women.The invention also relates to the in vitro use of somatostatin or one ofits agonist analogues in order to inhibit the start of growth ofquiescent follicles in ovarian tissue, or the in vitro use of asomatostatin antagonist analogue in order to accelerate the start ofgrowth of quiescent follicles in ovarian tissue.

BACKGROUND OF THE INVENTION

In women, as in all mammals, fertility is dependent on the presence inthe ovaries of female gametes called “oocytes”. In humans, the oocytecapital is constituted once and for all at birth; the number of oocytesis then comprised between 500 000 and 1 million per ovary. These oocytesare surrounded by a few granulosa cells; this functional group is calledan ovarian follicle (Gougeon, A., Endocrine Reviews (1996), 17,121-155). At birth, but also throughout life until menopause, themajority of the ovarian follicles are in a dormant state.

From its constitution, the oocyte capital progressively diminishes:thus, there are approximately 200 000 follicles per ovary at puberty,approximately 80 000 at 20 years of age, approximately 30 000 at 30years of age, approximately 10 000 at 40 years of age, the capital beingpractically depleted at around 50 years of age (cf. Gougeon, A. andLefevre, B., <<Folliculogenese et maturation ovocytaire>> in Medecine deis reproduction, 3.sup.rd edition, Ed. Flammarion, p. 49). The depletionof the oocyte capital corresponds clinically to menopause. The dormantfollicles present in the ovary at a given time constitute the “ovarianreserve”.

Two mechanisms are involved in the progressive depletion of the ovarianreserve. Approximately 80% of the follicles disappear at the start ofapoptosis, while the remaining 20% start to grow. The latter then begina long process of development (approximately 6 months) in which aminority of them (approximately 400 over a lifetime) will arrive at thestage of preovulatory follicles containing a mature oocyte which is ableto be to be fertilized (Gougeon, A., Endocrine Reviews (1996), 17,121-155). The majority of the growing follicles disappear throughapoptosis leading to their involution; apoptosis strikes them at anystage of their development.

The change from the quiescent follicle stage to the growing folliclestage is a phenomenon which is continuous but of variable intensity. Inparticular, it accelerates in the 10 to 15 years preceding menopause,from approximately 38 years of age.

The factors stimulating the first stages of growth (starting from thelarge primary follicle) are relatively well known. They includegonadotropins (LH and FSH) but particularly growth factors and steroidssuch as androgens. However, the mechanisms controlling the initiation offollicle growth are not well known. It is well established that thisstage of folliculogenesis is not dependent on gonadotropins (LH and FSH)(cf. for example Bullun, S. and Adashi, E., Williams Textbook ofEndocrinology, Tenth Edition (2003), 587-664). A hormone known as AMH(Anti-Mullerian hormone) could be involved in maintaining the quiescenceof the follicles while a peptide known as Kit-Ligand (also called SCF)could be involved in activating the growth of dormant follicles. Inaddition, a growth factor known as GDF-9 seems to be important formaintaining the growth once it is triggered.

Somatostatin (SST) is a cyclic peptide present in two forms in theorganism, one form containing 14 amino acids and one form containing 28amino acids. The biological activity of these two forms of SST issimilar. The SST-14 form is the predominant form in the central nervoussystem. It inhibits the secretion of the growth hormone by thesomatotrope cells of the anterior pituitary. The SST-28 form ispreferably expressed in the stomach and the pancreas. The biologicalactivity of SST is induced by means of a series of membrane receptorscoupled with a protein G, 5 sub-types of which have been characterized,namely the sub-types SSTR1 to SSTR5 (Reubi, J. C., Cancer Res., 47,551-558; Resine, T., et al., Endocr. Review, 16, 427-442; Lamberts, S W.et al., Endocr. Review, 12, 450-482).

The presence of SST in the ovary has been demonstrated in severalspecies including pigs (Mori, T. et al., Acta Endocrinol. (Copenh.)(1984), 106(2), 254-259), rats (McNeill, D. L. et al., Am. J. Anat.(1987), 179(3), 269-76) and in women (Holst et al., Hum. Reprod. (1994),9(8), 1448-1451). SST receptors have been identified in the ovary of therat (Lidor, A. et al., Gynecol. Endocrinol. (1998), 12(2), 97-101) aswell as in the human ovary in particular the sub-types 1, 2A and 5(Strauss et al., Hum. Reprod. (2003), 18, Suppl. 1, P-495).

The contribution of SST in ovarian physiology has been studied byseveral authors. In rats, the in vivo administration of SST seems toreduce the number of pituitary cells producing LH and FSH as well as thenumber of preovulatory follicles in the ovary (Nestorovic et al.,Histochem. J. (2001), 33(11-12), 695-702). In vitro, SST inhibitsaromatase and the production of progesterone, stimulated by FSH, in amodel of granulosa cells (Andreani, C. L. et al., Hum. Reprod. (1995),10(8), 1968-1973). In pigs, SST inhibits the increase in cAMP induced byLH and forskolin in the granulosa cells (Rajkumar, K. et al., J.Endocrinol. (1992), 134(2), 297-306), and seems to inhibit the nuclearmaturation of the preovulatory oocyte (Mori, T. et al., Acta Endocrinol.(Copenh.) (1985), 110(3), 408-412). In women, in vitro studies ongranulosa cells from preovulatory follicles suggest a direct role of SSTin inhibiting the synthesis of IGF-BP1 and of progesterone (Holst, N. etal., Fertil. Steril. (1997), 68(3), 478-482). In women, in vivo, SST iscapable of reducing the secretion of LH by the pituitary, reducing theproduction of androgens and the IGF-1 serum levels. By contrast, SSTincreases the serum levels of IGF-BP3 (Fulghesu, A. M. et al., Fertil.Steril. (1995), 64(4), 703-708; Piaditis, G. P. et al., Clin.Endocrinol. (Oxf.) (1996), 45(5), 595-604). SST was co-administered withFSH during treatment to induce ovulation in patients who are infertileas a result of a polycystic ovary syndrome. The capacity of SST toreduce the LH serum levels, and to reduce the serum levels of growthhormone and of IGF-I has been confirmed. This endocrine effect is nothowever accompanied by a significant impact on the follicle growthinduced by the administration of FSH (Lidor, A. et al., Gynecol.Endocrinol. (1998), 12(2), 97-101; van der Meer, M. et al., Hum. Reprod.(1998), 13(6), 1465-1469). In summary, until now a marginal effect ofSST on the pituitary secretion of LH and on the production ofprogesterone by the granulosa cells of preovulatory follicles has beenreported.

BRIEF SUMMARY OF THE INVENTION

The applicants have now discovered, in a surprising manner, that SST andits analogues have the capacity to inhibit the transition of thefollicles from a quiescent stage to a growth stage and that this facultyallows new therapeutic uses for these compounds.

The benefit of this discovery resides primarily in the possibility ofusing SST or an SST agonist analogue for preparing a medicament intendedto diminish or inhibit the start of growth of follicles in the quiescentstage. Secondly, it is also possible to use an antagonist analogue ofthis peptide in order to prepare a medicament intended to accelerate thestart of growth of the quiescent follicles.

There exists a set of clinical situations for which it would bedesirable from a medical perspective for the patient to slow the use ofthe ovarian reserve in order to delay the depletion of the latter andtherefore to preserve the ovarian function and fertility. Thesesituations are typically, and in a non-exclusive manner, patients atrisk of early menopause. It is well known that certain patients havepremature depletion of their follicular capital. Menopause then occursbefore 40 years of age and sometimes even before thirty years of age. Itis often possible to predict this early menopause on the basis of familyantecedents, or genetic anomalies such as Turner syndrome (complete orpartial). In this situation, the administration of SST or of one of itsagonist analogues is a preventive measure and aims to slow the start ofgrowth of the quiescent follicles.

The same applies for patients having difficulty conceiving and for whomthe chronological age or biological age of their ovaries corresponds tothe period of acceleration of activation of the quiescent follicles:slowing this depletion of follicular capital should make it possible toincrease the efficiency of the treatments and the to chances of becomingpregnant.

Another clinical situation which may benefit from a treatment by SST orby one of its agonist analogues is the graft (preferably an autograft)of an ovary or of ovary fragments. In this context, the resumption ofovarian function is often temporary and is accompanied by a prematuredepletion of the number of primordial follicles (Baird, is D. T. et al.,Endocrinology (1999), 140, 462-471). It has in fact been demonstratedthat, during the transplantation, the granulosa cells of the growingfollicles are more inclined to start apoptosis than those of theprimordial follicles (Liu, J. et al., Hum. Reprod. (2002), 17, 605-611).Moreover, the removal of ovarian tissue and its fragmentation causes theprimordial follicles to move rapidly and en masse toward a stage of lateprimary follicles (cf. Wandji S-A, et al., Hum. Reprod. (1997), 12,1993-2001; cf. also the control group of the example of the presentapplication).

In the context of this application, somatostatin agonist analogues (orsomatostatin) can be added to the various media for sampling, washing,preserving, freezing and thawing of ovarian tissue for the purpose ofgrafts. The invention therefore also relates to the corresponding mediacomprising a somatostatin agonist analogue. It also relates to the useof a somatostatin agonist analogue as a protective adjuvant for themedia for sampling, washing, preserving, freezing and thawing of ovariantissue.

Moreover, the in vitro use of somatostatin or of somatostatin agonistanalogues can also be useful in the field of toxicological analyses andin the in vitro production of mature oocytes from fresh or frozenovarian tissue. As regards the former, during tests of the effect of newchemical entities on ovarian follicles and their growth, the addition tothe follicle sample of a somatostatin agonist analogue makes it possibleto slow the follicle growth and thus to more easily observe any effectof acceleration of said growth caused by said new chemical entities. Theaddition of somatostatin antagonist makes it possible to move thefollicular reserve towards the growth phases and to better evaluate theimpact of new chemical entities on this phenomenon. As regards the invivo production of mature oocytes, the addition to the follicle sampleof a somatostatin agonist analogue allows the follicle growth to beslowed during the initial phase of culture. The addition of somatostatinantagonist in a second phase subsequently allows the follicular reserveto be moved toward the growth phases allowing a greater number of maturefollicles and therefore of fertilizable oocytes to be obtained.

The use of somatostatin agonist analogues (or of somatostatin) inpatients with polycystic ovaries is also beneficial. In fact, numerousobservations suggest that polycystic ovaries have an abnormally highfollicle population (cf. Hughesdon, Obstet. to Gynecol. Surv. (1982),37(2), 59-77). The excessive production of androgens by these folliclesin an excessive quantity could be the origin of the metabolic andendocrine disorders observed in these patients. Moreover, a significantreduction of this follicle population by ovarian resection,cauterization or ultraviolet radiation, constitutes one of the mosteffective therapies because it allows the patients to ovulate atapproximately 80% and to become pregnant at a cumulative rate of 75%over 18 months. Thus, the regular administration of a somatostatinagonist analogue (or of somatostatin) should bring about a reduction inthe number of growing follicles and therefore of the supernumeraryantral follicles producing androgens resulting in the resumption offertile ovulatory cycles of a temporary or permanent nature.

Moreover, the use of somatostatin agonist analogues (or of somatostatin)in patients who are about to have, are currently having or have hadchemotherapy or irradiation (for therapeutic or other purposes) reducesthe risk of early menopause by preventing the accelerated activation ofthe follicular reserve which would make it more sensitive to thechemotherapy agents or to the ionizing radiation.

Other applications can also be envisaged, in particular in theveterinary field. The invention could be used to save species, the useof somatostatin agonist analogues (or of somatostatin) allowing theovarian reserve of females to be preserved. Similarly, somatostatinantagonist analogues can be used in the context of in vitro or in vivofertilizations in animals of high commercial value. Such animals withhigh commercial value can in particular be horses, bovines, ovines orgoats; they can also be animals of transgenic origin.

In addition to the pathologies mentioned above, a systematic slowing ofthe depletion of the ovarian reserve could be envisaged in women notsuffering from any ovarian dysfunction. In industrialized countries, thecontinuous extension of life expectancy (currently approximately 83years in France) is accompanied by an extension of the post-menopausalperiod and of the problems associated with it: cardiopathies,osteoporosis, cutaneous aging, etc. Doubts are raised as to thelong-term safety of hormonal substitute treatments for menopause. Anattractive alternative would consequently be delaying the age at whichmenopause occurs. This would thus reduce the post-menopausal period andthe associated risks. This delay would not however mean that fertilitycould be maintained up to 60 plus years of age. Numerous works suggestthat ovarian function is maintained as long as a minimum number offollicles of the reserve is maintained, and that despite a “normal”ovarian function (steroid levels barely affected), the chances ofpregnancy are extremely low.

In situations where it is sought to slow the use of the ovarian reserve,according to the invention natural somatostatin (SST14 or SST28), or,preferably, a somatostatin agonist analogue (natural or synthetic), willbe used. The somatostatin agonist analogue can be a cyclic or non-cyclicpolypeptide, a fusion or recombination protein, a non-peptidic chemicalentity (i.e. peptidomimetic) or also an “SS-like” peptide such ascorticostatin. The agonist analogues to be used must have high affinityfor the SST receptor and induce a functional activity thereof such asthe inhibition of the secretion of growth hormone by pituitarysomatotrope cells and/or the inhibition of the in vitro proliferation ofpituitary adenoma cells. Preferably, the somatostatin agonist analoguehas high affinity for all or at least 2 or 3 of the sub-types of SSTreceptors or a greater affinity for at least one of the sub-types 1, 2,3, 4 and 5 (for example for sub-type 2).

Agonist analogues of somatostatin have been described in particular inthe patent application PCT WO 01/00676 or WO 98/08528 or also in theU.S. Pat. Nos. 6,387,932, 6,268,342, 6,057,338, 6,025,372.

DETAILED DESCRIPTION OF THE INVENTION

According to a particular variant of the invention, the somatostatinagonist analogues are compounds of general formula (I)

in which:

X₁ is a radical of formula (a) or (b)

R₁ independently representing each time it occurs an optionallysubstituted phenyl radical in which the optional substituents areindependently chosen from a halogen atom and the methyl, ethyl, methoxyand ethoxy radicals,

R₂ representing —Z₁—CH₂—R₁, —CH₂—CO—O—CH₂—R₁,

Z₁ being O or S;

X₂ is an α-amino acid having an aromatic residue on the side chainC_(α), or an amino acid unit chosen from Dab, Dpr, Dpm, His, (Bzl)HyPro,thienyl-Ala, cyclohexyl-Ala and t-butyl-Ala;

A is a divalent residue chosen from Pro,

R₃ is NR₈R₉—C₂₋₆alkylene, guanidino-C₂₋₆alkylene or C₂₋₆alkylene-COOH,R_(3a) is H, C₁₋₄alkyl or has, independently, one of the meanings givenfor R₃, R_(3b) is H or C₁₋₄ alkyl, R_(a) is OH or NR₅R₆, R_(b) is—(CH₂)₁₋₃— or —CH(CH₃)—, R₄ is H or CH₃, R_(4a) is benzyl optionallysubstituted on the aromatic ring, each of R₅ and R₆ is independently H,C₁₋₄alkyl, ω-amino-C₁₋₄alkylene, ω-hydroxy-C₁₋₄alkylene or acyl, R₇ is adirect bond or C₁₋₆alkylene, each of R₈ and R₉ is independently H,C₁₋₄alkyl, ω-hydroxy-C₂₋₄alkylene, acyl or CH₂OH—(CHOH)_(c)—CH₂— inwhich c is 0, 1, 2, 3 or 4, or R₈ and R₉ form together with the nitrogenatom to which they are attached a heterocyclic group which can includean additional heteroatom, and R₁₁ is benzyl optionally substituted onthe aromatic ring, —(CH₂)₁₋₃—OH, CH₃—CH(OH)— or —(CH₂)₁₋₅—NR₅R₆, andZZ_(a) is a natural or unnatural α-amino acid unit;

it being understood that X₁, X₂ and Lys each have the configuration L;or are pharmaceutically acceptable salts or protected forms of compoundsof general formula (I).ZZ_(a) can have a configuration D or L. ZZ_(a) can be for example Thr,Ser, Ala, Val, Ile, Leu, Nle, His, Arg, Lys, Nal, Pal, Tyr, Trp, Phesubstituted on the aromatic ring or N^(α)-benzyl-Gly. When ZZ_(a) isPhe, its benzene ring can be substituted for example by NH₂, NO₂, CH₃,OCH₃ or a halogen atom, preferably in position para. When ZZ_(a) is Phe,its benzene ring is preferably not substituted.

When A comprises a Pro amino acid residue, any substituent present onthe proline ring, for example R₃—NH—CO—O— etc., is preferably inposition 4. Such substituted proline residues can be in the cis form,for example

such as in the trans form. Each geometric isomer individually as well asmixtures of these isomers are included in the uses according to theinvention. When A is

in which NR₈R₉ forms a heterocyclic group, said group can be aromatic orsaturated and can include a nitrogen atom or a nitrogen atom and asecond hetero atom chosen from nitrogen and oxygen. Preferably, theheterocyclic group is for example pyridyl or morpholino. TheC₂₋₆alkylene radical in this residue is preferably —CH₂—CH₂—.

An acyl group such as R₅, R₆, R₈ and R₉ in A can for example be anR₁₂CO— group in which R₁₂ is H, C₁₋₄alkyl, C₂₋₄alkenyl, C₃₋₆cycloalkylor benzyl, and methyl or ethyl.

When R_(4a) or R₁₁ in A is benzyl substituted on the aromatic ring, thebenzene ring can be substituted as indicated above for ZZ_(a).

According to a preferred variant of the invention, the somatostatinagonist analogues are compounds of general formula (II)

in which R is NR₁₀R₁₁—C₂₋₆alkylene or guanidine-C₂₋₆alkylene, and eachof R₁₀ and R₁₁ is independently H or C₁₋₄alkylor are pharmaceutically acceptable salts or protected forms of compoundsof general formula (II).

Preferably, R is NR₁₀R₁₁—C₂₋₆alkylene. The preferred compounds ofgeneral formula (II) are those such that R is 2-aminoethyl (and inparticular the peptide SOM 230 of the formulacyclo[{4-(NH₂—C₂H₄—NH—CO—O—)Pro}-Phg-DTrp-Lys-Tyr(4-Bzl)-Phe] thestructure of which is reproduced below).

By “protected form” of a compound of general formula (I) or (II), ismeant in the present application a somatostatin analogue in which atleast one of the amino groups is protected and the deprotection of which(which preferably is itself carried out in physiological medium) leadsto a compound of general formula (I) or (II). Suitable protective groupsfor amino groups are for example those described in Protective Groups InOrganic Synthesis, T. W. Greene, 3. Wiley & Sons NY (1981), 219-287. Anexample of such a protective group for an amino group is the acetylgroup.

Among the somatostatin agonist analogues which can be used according tothe invention, lanreotide, octreotide, vapreotide, SOM 230 (seestructure below), MK-678 (peptide of structurecyclo(N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe)), BIM-23190 (peptide of structureN-hydroxyethylpiperazinyl-acetyl-D-Phe-cyclo[Cys-Tyr-D-Trp-Lys-Abu-Cys]-T-hr-NH₂),BIM-23197 (peptide of structureHepes-D-Phe-cyclo[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH₂ in which Aburepresents aminobutyric acid), BIM-23268 (peptide of structurecyclo[Cys-Phe-Phe-D-Trp-Lys-Thr-Phe-Cys]-NH₂), PTR-3173 (see structurebelow), TT-232 (of structure D-Phe-cyclo[Cys-D-Trp-Lys-Cys]-Thr-NH₂),and their pharmaceutically acceptable salts can more particularly bementioned; the synthetic peptide of formula c[Tic-Tyr-DTrp-Lys-Abu-Phe]and its pharmaceutically acceptable salts can also be mentioned; finallythe KE 108 peptide of formulaTyr⁰-(cyclo-D-Dab-Arg-Phe-Phe-D-Trp-Lys-Thr-Phe) described in particularin Reubi et al., Eur. J. Pharmacol. (2002), 456, 45-49, can bementioned. The use of lanreotide, octreotide or one of theirpharmaceutically acceptable salts, and more particularly lanreotide orone of its pharmaceutically acceptable salts is quite particularlypreferred.

In a similar way to the compounds of general formula (I) or (II), theabove-mentioned peptides can also be presented in a protected form. Thedefinition of the protected form given above for the compounds ofgeneral formulae (I) or (II) is applicable mutatis mutandis.

According to a preferred variant of the invention, the patients for whomthe medicament based on somatostatin or a somatostatin agonist analoguementioned above is intended are women having an early menopause riskfactor, and in particular women with a family history of earlymenopause. According to a particular variant of the invention, thepatients for whom the medicament based on somatostatin or somatostatinagonist analogue mentioned above is intended are women who have an Xchromosome microdeletion or a partial Turner syndrome.

The second benefit of the discovery mentioned above resides in thepossibility of preparing a medicament based on a somatostatin antagonistanalogue in order to accelerate the start of growth of the quiescentfollicles. In fact, one couple in six of those who wish to achieve apregnancy has difficulty conceiving. Although there are many causes, twotypes of treatment have emerged and are commonly used in human medicinefor the treatment of sterility. These treatments, also called “MedicallyAssisted Procreation” (MAP), consist firstly of inducing thesimultaneous growth of several preovulatory follicles. This makes itpossible to obtain several mature oocytes, and therefore severalembryos, and thus to increase the chances of conception. This isachieved by the administration of one or more medicaments stimulatingthe pituitary secretion of gonadotropins (FSH and LH), such as ananti-estrogen (for example clomiphene citrate or tamoxifen) or anaromatase inhibitor (for example letrozole, anastrazole or exemestane).The simultaneous growth of several preovulatory follicles can also beinduced by the administration of a preparation of human FSH (extractiveor recombinant) combined or not combined with LH. When the follicleshave reached a preovulatory size, depending on the cause of sterility,two treatment options exist. The first is to carry out an intrauterineinsemination (IUI) and the second is to remove the oocytes from theovary by aspiration of the follicles (between 5 and 15 oocytes) and tocarry out an insemination in the laboratory (in vitro), either by simplecoincubation of the oocytes with the partner's sperm (IVF) or bymicroinjection of sperm directly into the oocyte (ICSI). It is essentialto obtain several mature oocytes in order to optimize the success rates(pregnancy rates) obtained with these treatments; however in certainwomen, despite appropriate ovarian stimulation, the number of oocytesobtained is low or even equal to one. This difficulty in responding tothe stimulating treatment is a result of the limited number of growingfollicles present in the ovaries of these patients. It is therefore ofconsiderable therapeutic benefit to be able to activate follicles of theovarian reserve and make them enter the growth phase.

Another subject of the present invention is the use of a somatostatinantagonist analogue for preparing a medicament intended to acceleratethe start of growth of the quiescent follicles in non-menopausal women.

The administration of such a medicament over a period of 1 to 12 monthsin women leads to an increase in the number of follicles in the growthphase and which are therefore able to be stimulated with the standardtreatments in order to reach the stage of preovulatory follicles.

Another application of the ability of the somatostatin antagonistanalogues to induce early follicle growth is their in vitro use infollicle cultures for the production of mature to oocytes intended forfertilization. The somatostatin antagonist analogue is added to theculture media used to support in vitro follicle development. Theinvention therefore also relates to the corresponding media comprising asomatostatin antagonist analogue. Moreover, the ability of thesomatostatin antagonist analogues to induce early follicle growth canalso be used in the field of toxicological analyses. In particular,during tests of the effect of new chemical entities on follicle growth,the addition to the follicle sample of a somatostatin antagonistanalogue makes it possible to accelerate follicle growth and thus tomore easily observe any effect of slowing of said growth caused by saidnovel chemical entities.

The somatostatin antagonist analogue can be a cyclic or non cyclicpolypeptide, a fusion or recombination protein, a non-peptide chemicalentity (i.e. a peptidomimetic) or also a “SS-like” peptide such ascorticostatin. The antagonist analogues to be used must have a highaffinity for the SST receptor and inhibit the functional activity ofSST14 or SST28 such as the inhibition of the secretion of growth hormoneby somatotrope cells of the pituitary and/or the inhibition of the invitro proliferation of pituitary adenoma cells. Preferably, thesomatostatin antagonist analogue has a high affinity for all or at least2 or 3 of the sub-types of SST receptors or a greater affinity for atleast one of the sub-types 1, 2, 3, 4 and 5 (for example for sub-type2).

A somatostatin antagonist analogue which can be used for the preparationaccording to the invention can for example be a peptide of generalformula

A¹-cyclo{D-Cys-A²-D-Trp-A³-A⁴-Cys}-A⁵-Y¹  (III)

in which:

A¹ is an optionally substituted aromatic α-amino acid;

A² is an optionally substituted aromatic α-amino acid;

A³ is Dab, Dap, Lys or Orn;

A⁴ is β-Hydroxyvaline, Ser, Hser, or Thr;

A⁵ is an optionally substituted aromatic D- or L-α-amino acid; and

Y¹ is OH, NH₂ or NHR¹, R¹ being (C₁₋₆)alkyl;

each optionally substituted aromatic α-amino acid being optionallysubstituted with one or more substituents independently chosen from thegroup comprising a halogen atom and the groups NO₂, OH, CN, (C₁₋₆)alkyl,(C₂₋₆)alkenyl, (C₂₋₆)alkynyl, (C₁₋₆)alkoxy, Bzl, O-Bzl and NR⁹R¹⁰, R⁹and R¹⁰ each being independently H, O, or (C₁₋₆) alkyl; andeach nitrogen atom with a peptide amide bond and the amino group of A¹being optionally substituted with a methyl group, it being understoodthat there is at least one such methyl group in a peptide of generalformula (III);or a pharmaceutically acceptable salt of a peptide of general formula(III).

By “aromatic α-amino acid” is meant an amino acid residue of formula

in which Z₁ is a radical containing an aromatic ring and Z₂ is ahydrogen atom or a radical containing an aromatic ring. Examples of suchradicals containing an aromatic ring include, but are not limited to, abenzene or pyridine ring and the following structures with or withoutone or more X substituents on the aromatic ring (X being, independentlyeach time that it occurs, a halogen atom, NO₂, CH₃, OCH₃, CF₃ or OH):

Other examples of an “aromatic α-amino acid” according to the inventionare substituted His, such as MeHis, His (τ-Me) or His (π-Me).

Other somatostatin antagonist analogues have been described inparticular in the patent applications PCT WO 98/08528, WO 98/08529, WO98/24807, WO 98/44921, WO 98/44922, WO 98/45285 and WO 99/22735, or alsoin the U.S. Pat. Nos. 6,387,932, 6,262,229, 6,063,796, 6,057,338,6,025,372, 5,925,618, 5,846,934 and 4,508,711.

Among the somatostatin antagonist analogues which can be used accordingto the invention and their pharmaceutically acceptable salts, there maymore particularly be mentioned:

-   -   the following peptides of general formula (III):

-   Cpa-cyclo[D-Cys-Pal-D-Trp-N-Me-Lys-Thr-Cys]-D-Trp-NH₂;

-   Cpa-cyclo[D-Cys-Tyr-D-Trp-N-Me-Lys-Thr-Cys]-Nal-NH₂;

-   Cpa-cyclo[D-Cys-Pal-D-Trp-N-Me-Lys-Thr-Cys]-Nal-NH₂;    -   the peptide known by the code name AC-178,335 (of structure        acetyl-D-His-D-Phe-D-Ile-D-Arg-D-Trp-D-Phe-NH₂);    -   the octapeptide known by the code name ODN-8 (cf. FIG. 1 of        Proc. Natl. Acad. Sci. USA (2000), 97(25), 13973-13978);

-   -   the peptide known by the code name SB-710411 (of structure        Cpa-cyclo[D-Cys-Pal-D-Trp-Lys-Val-Cys]-Cpa-amide);    -   the peptide known by the code name BIM-23056 (of the structure        represented below);    -   the compound known by the code name BN-81674 (of the structure        represented below);    -   the compound known by the code name SRA-880 (of the structure        represented below);        and their pharmaceutically acceptable salts.

In a similar way to the compounds of general formula (I) or (II), theabove-mentioned peptides (including those corresponding to generalformula (III)) can also be presented in a protected form. The definitionof the protected form given above for the compounds of general formulae(I) or (II) is applicable mutatis mutandis.

By somatostatin agonist analogue is meant in the present application acompound for which the effective dose DE₅₀ determined in the test of theagonist effect described below is less than or equal to 1 μM for atleast one of the somatostatin sub-receptors.

By somatostatin antagonist analogue is meant in the present applicationa compound for which the effective dose DE₅₀ determined in the test ofthe antagonist effect described below is less than or equal to 1 μM forat least one of the somatostatin sub-receptors.

By pharmaceutically acceptable salt is meant in particular in thepresent application addition salts with inorganic acids such ashydrochloride, hydrobromide, hydroiodide, sulphate, phosphate,diphosphate and nitrate or with organic acids such as acetate, maleate,fumarate, tartrate, succinate, citrate, lactate, methanesulphonate,p-toluenesulphonate, pamoate and stearate. Also included in the field ofthe present invention, when they can be used, the salts formed frombases such as sodium or potassium hydroxide. For other examples ofpharmaceutically acceptable salts, reference can be made to “Saltselection for basic drugs”, Int. J. Pharm. (1986), 33, 201-217.

According to the present invention, the pharmaceutical preparationscontaining somatostatin or one of its agonist or antagonist analoguesapplicable in this invention can be administered by parenteral route(subcutaneous, intramuscular, intraperitoneal, intravenous, or in animplant), by oral, vaginal, rectal, nasal, sublingual or transdermalroute. The vaginal route is preferred because it allows effectiveconcentrations of the active ingredient to be delivered to the ovarywhile minimizing systemic exposure. The somatostatin or the somatostatinanalogue used is formulated with the necessary excipients known to aperson skilled in the art, in order to allow an effective andreproducible administration for each administration route.

The dose of a product according to the present invention, intended forthe treatment of the above-mentioned diseases or problems, variesaccording to the method of administration, the age and body weight ofthe subject to be treated as well as the condition of the subject, andthe final decision is made by the attending doctor or vet. Such aquantity determined by the attending doctor or vet is called“therapeutically effective quantity” here.

The following typical situations for a use according to the inventioncould however be envisaged:

-   -   A patient of approximately 20 to 25 years of age (for example)        has a partial Turner syndrome through X chromosome        microdeletion. Her ovarian function is apparently normal with        regular, ovulatory cycles. Her FSH serum level is slightly        higher during the luteal-follicular transition period (for        example FSH=approximately 9.2 IU/litre). The ovarian ultrasound        carried out by trans-vaginal route shows ovaries of normal        volume with a slightly reduced number of antral follicles.        Considering the high risk that she will have early menopause,        the patient is treated with lanreotide acetate at a dose of 120        mg/months (Somatuline® Autogel® 120 mg, Beaufour Ipsen Pharma,        France). The treatment is discontinued after several years when        the patient wishes to conceive.    -   A patient of approximately 35 to 40 years of age has had primary        sterility for several years. Assessment of the couple produced a        diagnosis of sterility of tubal origin, very probably resulting        from a history of peritonitis. The menstrual cycles are        ovulatory and the FSH serum level is slightly higher during the        luteal-follicular transition period (for example        FSH=approximately 11.4 IU/L). The ovarian ultrasound carried out        by trans-vaginal route shows ovaries with a slightly reduced        volume with a reduced number of antral follicles (approximately        3 per ovary). A diagnosis of reduction of the ovarian reserve is        made. An in vitro fertilization treatment is recommended and the        patient undergoes ovarian stimulation treatment with daily        injection of 225 units of recombinant FSH. On the 6^(th) day of        stimulation, an ovarian ultrasound shows a single growing        follicle of 14 mm in the right ovary. The dose of FSH is doubled        and the patient is seen again 2 days later. A single 18-mm        follicle is observed, which confirms a reduction in the ovarian        reserve. The treatment is discontinued. After return of a        spontaneous cycle, a treatment by daily administration of a        somatostatin antagonist analogue is initiated. During this to        treatment, the number of antral follicles present in each ovary        is assessed by ultrasound at the start of each menstrual cycle.        After 4 months of treatment, the number of antral follicles is        on average approximately 6 per ovary and the serum FSH has been        reduced. A stimulation by recombinant FSH is initiated, multiple        follicular development is obtained, and a standard in vitro        fertilization procedure is carried out.    -   A patient with polycystic ovary syndrome has very irregular        periods, absence of ovulation, excessive weight and cutaneous        signs of androgen excess such as acne and hirsutism. When the        pelvis is examined by ultrasound, the endometrium is        hyperplastic, the ovaries have increased volume, an increased        stroma, and more than 10 antral follicles per ovarian section.        No follicle has a diameter greater than 10 mm. The patient is        treated with lanreotide acetate at a dose of 120 mg/month        (Somatuline® Autogel® 120 mg, Beaufour Ipsen Pharma, France).        After 3 months of treatment, the patient has spontaneous        periods. In the 4^(th) month of treatment, the ultrasound of the        ovaries indicates a reduction in ovarian volume and in the        number of antral follicles. One 16 mm follicle is observed.        During the 5^(th) and 6^(th) months of treatment, the patient        has regular periods and her temperature curve is biphasic,        suggesting an ovulation. In the 8^(th) month of treatment, the        patient does not have a period and a pregnancy test is positive.        The treatment with lanreotide acetate is discontinued.

PARTICULAR ABBREVIATIONS AND DEFINITIONS USED IN THE PRESENT APPLICATION

The abbreviations of the common amino acids are in accordance with theIUPAC-IUB recommendations. Moreover, the definitions for certainabbreviations used in the present application are as follows:

-   -   Abu=α-aminobutyric acid;    -   Aib=α-aminoisobutyric acid;    -   β-Ala=β-alanine;    -   Amp=4-aminophenylalanine;    -   Ava=5-aminovaleric acid;    -   Bzl=benzyl;    -   Cha=cyclohexylalanine;    -   Cpa=3-(4-chlorophenyl)alanine;    -   Dab=2,4-diaminobutyric acid;    -   Dap=2,3-diaminopropionic acid;    -   Dip=3,3′-diphenylalanine;    -   GABA=γ-aminobutyric acid;    -   HSer=homoserine;    -   1-Nal=3-(1-naphthyl)alanine;    -   2-Nal=3-(2-naphthyl)alanine;    -   Nle=norleucine;    -   Nva=norvaline;    -   2-Pal=3-(2-pyridyl)alanine;    -   3-Pal=3-(3-pyridyl)alanine;    -   4-Pal=3-(4-pyridyl)alanine;    -   Phg=—HN—CH(C₆H₅)—CO—    -   Tfm=trifluoromethyl;    -   TfmA=4-trifluoromethylphenyl-alanine;    -   Tic=1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.

Moreover, NMeLys represents the N-methyl-lysine, in which the nitrogenof the peptide bond is methylated (and not the nitrogen of the sidechain of the lysine).

Finally, Tyr(I) represents an iodized tyrosine residue (for example3-I-Tyr, 5-I-Tyr, 3,5-I-Tyr) in which the iodine atom can be aradioactive isotope, for example I₁₂₅, I₁₂₇ or I₁₃₁.

Moreover, the term “approximately” refers to an interval around thevalue considered. As used in the present application, “approximately X”means an interval of X less 10% of X to X plus 10% of X, and preferablyan interval of X less 5% of X to X plus 5% of X.

Preparation of the Peptides of General Formula (I):

The peptides of general formulae (I) and (II) mentioned above and theirsynthesis are described for instance in the patent applications PCT WO97/01579 and WO 02/10192.

The peptides of general formula (III) mentioned above and theirsynthesis are described in the patent application PCT WO 02/072602.

Unless they are otherwise defined, all the technical and scientificterms used here have the same meaning as that usually understood by anordinary specialist in the field to which this invention belongs.Similarly, all the publications, patent applications, all the patentsand all other references mentioned here are incorporated by way ofreference.

The following examples are given in order to illustrate the aboveprocedures and must in no event be considered to be a limit to the scopeof the invention.

EXAMPLES Example 1

Ovaries of adult ewes are collected immediately after slaughter. Theovaries are placed in an organ transport medium without serum (X-vivo,Bio Whittaker, Walkersville, Md., USA) at 10° C. and transported to thelaboratory. Approximately 1 h after removal, the cortex is isolated fromthe medulla then fractionated into slices of 2 mm thickness (1 cm²,average weight of 212 mg) after rinsing in new X-vivo. The cortexfragments are cultured in an oven under 5% oxygen for 10 days in wellplates in the presence of DMEM. The medium is changed every 2 days.

In the control fragments (incubated in the absence of SST) theprimordial follicles gradually progress to the state of follicles at thestart of growth (see FIGS. 1 and 2). The addition of SST14 atconcentrations varying between 10⁻⁹ M and 10⁻⁶ M very significantlyinhibits the start of growth of the primordial follicles as is shown bythe maintenance over time of the number of primordial follicles (cf.FIG. 1) and the absence of increase in the number of primary follicles(cf. FIG. 2).

Example 2

The procedure used is the same as for Example 1, except that thesomatostatin is replaced with one of its agonist analogues, namely thesynthetic peptide of formula c[Tic-Tyr-DTrp-Lys-Abu-Phe] (hereafter AG₁peptide).

In the control fragments (incubated in the absence of AG₁ peptide) theprimordial follicles gradually progress to the state of follicles at thestart of growth (cf. FIG. 3), The addition of AG₁ peptide at aconcentration of 10⁻⁹M very significantly inhibits the start of growthof the primordial follicles as is shown by the maintenance over time ofthe number of primordial follicles and the absence of increase in thenumber of primary follicles (cf. FIG. 4).

Example 3

The procedure used is the same as for Example 1, except that thesomatostatin is replaced by one of its antagonist analogues, namely thesynthetic peptide of formulaCpa-c(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂ (hereafter ANT₁peptide).

In the control fragments (incubated in the absence of ANT₁ peptide) theprimordial follicles gradually progress to the state of follicles at thestart of growth (cf. FIG. 3). It is observed that the addition of ANT₁peptide at a concentration of 10⁻⁶M accentuates the start of growth ofthe primordial follicles (cf. FIG. 5).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents the proportions of dormant follicles during a periodof 10 days of culture of an ovarian cortex of a ewe in the presence orabsence (control) of somatostatin (SST14). These proportions aremeasured for each sample tested on the day of the start of theexperiment (D0), on the 4^(th) day (D4), on the 7^(th) day (D7) and onthe 10^(th) day (D10).

FIG. 2 represents the proportions of primary follicles during a periodof 10 days of culture of an ovarian cortex of a ewe in the presence orabsence (control) of somatostatin (SST14). These proportions aremeasured for each sample tested on the to day of the start of theexperiment (D0), on the 4^(th) day (D4), on the 7^(th) day (D7) and onthe 10^(th) day (D10).

FIG. 3 represents the proportions of primordial, intermediate, primaryand secondary follicles during a period of 10 days of culture of anovarian cortex of a ewe in the absence of agonist or somatostatinantagonist analogue. These proportions are measured for each sampletested on the day of the start of the experiment (D0), on the 4^(th) day(D4), on the 7^(th) day (D7) and on the 10^(th) day (D10).

FIG. 4 represents the proportions of primordial, intermediate, primaryand secondary follicles during a period of 10 days of culture of anovarian cortex of a ewe in the presence or absence of a somatostatinagonist analogue, the synthetic peptide of formulac[Tic-Tyr-DTrp-Lys-Abu-Phe] (AG₁ peptide). These proportions aremeasured for each sample tested on the day of the start of theexperiment (D0), on the 4^(th) day (D4), on the 7^(th) day (D7) and onthe 10^(th) day (D10).

FIG. 5 represents the proportions of primordial, intermediate, primaryand secondary follicles during a period of 10 days of culture of anovarian cortex of a ewe in the presence or absence of a somatostatinantagonist analogue, the synthetic peptide of formulaCpa-c(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂ (ANT₁ peptide). Theseproportions are measured for each sample tested on the day of the startof the experiment (D0), on the 4^(th) day (D4), on the 7^(th) day (D7)and on the 10^(th) day (D10).

TESTS FOR DETERMINATION OF THE MONIST OR ANTAGONIST EFFECT OF ASOMATOSTATIN ANALOGUE

Inhibition of the intracellular production of cAMP

CHO-K1 cells expressing the sub-types of human somatostatin (SRIF-14)receptors are cultured in 24-well plates in an RPMI 1640 mediumcontaining 10% foetal calf serum. The medium is changed the day beforethe experiment.

The cells at a rate of 10⁵ cells/well are washed twice with 0.5 ml ofnew RPMI medium comprising 0.2% BSA completed with 0.5 mM of3-isobutyl-1-methylxanthine (IBMX) and incubated for approximately 5minutes at approximately 37° C. The production of cyclic AMP isstimulated by the addition of 1 mM of forskolin (FSK; supplier: SigmaChemical Co., St. Louis, Mo., USA) for 15-30 minutes at approximately37° C.

Determination of the Agonist Effect of a Somatostatin Analogue

The agonist effect of a somatostatin analogue is measured by thesimultaneous addition of FSK (1 μM) and the analogue to be tested (10⁻¹⁰M to 10⁻⁵ M).

The reaction medium is eliminated and 200 ml of HCl 0.1 N are added. Thequantity of cAMP is measured by a radioimmunoassay (Kit FlashPlateSMP001A, New England Nuclear, Boston, USA).

Determination of the Antagonist Effect of a Somatostatin Analogue

The antagonist effect of a somatostatin analogue is measured by thesimultaneous addition of FSK (1 μM), SRIF-14 (1 to 10 nM) (supplier:Bachem, Torrence, Calif., USA) and the analogue to be tested (10⁻¹⁰ M to10⁻⁵ M).

The reaction medium is eliminated and 200 ml of HCl 0.1 N are added. Thequantity of cAMP is measured by a radioimmunoassay (Kit FlashPlateSMP001A, New England Nuclear, Boston, USA).

1. Use of somatostatin or one of its agonist analogues for preparing amedicament intended to regulate the ovarian follicular reserve, and inparticular to reduce the depletion of the ovarian follicular reserveover time, in non-menopausal women.
 2. Use according to claim 1,characterized in that somatostatin is used for preparing the medicament.3. Use according to claim 1, characterized in that a somatostatinagonist analogue is used for preparing of the medicament.
 4. Useaccording to claim 3, characterized in that the somatostatin agonistanalogue is a compound of general formula (I)

in which: X₁ is a radical of formula (a) or (b)

R₁ independently representing at each time that it occurs an optionallysubstituted phenyl radical in which the optional substituents areindependently chosen from a halogen atom and the methyl, ethyl, methoxyand ethoxy radicals, R₂ representing —Z₁—CH₂—R₁, —CH₂—CO—O—CH₂—R₁,

Z₁ being O or S; X₂ is an α-amino acid having an aromatic residue on theside chain C_(α), or an amino acid unit chosen from Dab, Dpr, Dpm, His,(Bzl)HyPro, thienyl-Ala, cyclohexyl-Ala and t-butyl-Ala; A is a divalentresidue chosen from Pro,

R₃ is NR₈R₉—C₂₋₆alkylene, guanidino-C₂₋₆alkylene or C₂₋₆alkylene-COOH,R_(3a) is H, C₁₋₄alkyl or has, independently, one of the meanings givenfor R₃, R_(3b) is H or C₁₋₄ alkyl, R_(a) is OH or NR₅R₆, R_(b) is—(CH₂)₁₋₃— or —CH(CH₃)—, R₄ is H or CH₃, R_(4a) is benzyl optionallysubstituted on the aromatic ring, each of R₅ and R₆ is independently H,C₁₋₄alkyl, ω-amino-C₁₋₄alkylene, ω-hydroxy-C₁₋₄alkylene or acyl, R₇ is adirect bond or C₁₋₆alkylene, each of R₈ and R₉ is independently H,C₁₋₄alkyl, ω-hydroxy-C₂₋₄alkylene, acyl or CH₂OH—(CHOH)_(c)—CH₂— inwhich c is 0, 1, 2, 3 or 4, or R₈ and R₉ form together with the nitrogenatom to which they are attached a heterocyclic group which can includean additional heteroatom, and R₁₁ is benzyl optionally substituted onthe aromatic ring, —(CH₂)₁₋₃—OH, CH₃—CH(OH)— or —(CH₂)₁₋₅—NR₅R₆, andZZ_(a) is a natural or unnatural α-amino acid unit; it being understoodthat X₁, X₂ and Lys each have the configuration L; or is apharmaceutically acceptable salt or protected form of a compound ofgeneral formula (I).
 5. Use according to claim 3, characterized in thatthe somatostatin agonist analogue is a compound of general formula (II)

in which R is NR₁₀R₁₁—C₂₋₆alkylene or guanidine-C₂₋₆alkylene, and eachof R₁₀ and R₁₁ is independently H or C₁₋₄alkyl or is a pharmaceuticallyacceptable salts or a protected form of a compound of general formula(II).
 6. Use according to claim 3, characterized in that thesomatostatin agonist analogue is chosen from the group comprisinglanreotide, octreotide, vapreotide, SOM 230, MK-678, BIM-23190,BIM-23197, BIM-23268, PTR-3173, TT-232, the peptide of formulac[Tic-Tyr-DTrp-Lys-Abu-Phe], the KE 108 peptide of formulaTyr⁰-(cyclo-D-Dab-Arg-Phe-Phe-D-Trp-Lys-Thr-Phe) and theirpharmaceutically acceptable salts and protected forms.
 7. Use accordingto claim 6, characterized in that the somatostatin agonist analogue islanreotide or one of its pharmaceutically acceptable salts.
 8. Useaccording to one of claims 1 to 7, characterized in that the medicamentis intended to be administered to a woman at risk of early menopause. 9.Use according to one of claims 1 to 7, characterized in that themedicament is intended to be administered to a woman who has an Xchromosome microdeletion.
 10. Use according to one of claims 1 to 7,characterized in that the medicament is intended to be administered to awoman who has polycystic ovaries.
 11. Use according to one of claims 1to 7, characterized in that the medicament is intended to beadministered to a woman who is about to have, is currently having or hashad chemotherapy or irradiation.
 12. Use of somatostatin or one of itsagonist analogues in toxicology tests relating to another compound inorder to determine the presence or the absence of an effect ofacceleration of follicle growth caused by said other compound.
 13. Useof a somatostatin antagonist analogue for preparing a medicamentintended to accelerate the start of growth of the quiescent follicles innon-menopausal women.
 14. Use according to claim 13, characterized inthat the somatostatin antagonist analogue is chosen from the peptides ofgeneral formula (III)A¹-cyclo{D-Cys-A²-D-Trp-A³-A⁴-Cys}-A⁵-Y¹  (III) in which: A¹ is anoptionally substituted aromatic α-amino acid; A² is an optionallysubstituted aromatic α-amino acid; A³ is Dab, Dap, Lys or Orn; A⁴ isβ-Hydroxyvaline, Ser, Hser, or Thr; A⁵ is an optionally substitutedaromatic D- or L-α-amino acid; and Y¹ is OH, NH₂ or NHR¹, R¹ being(C₁₋₆)alkyl; each optionally substituted aromatic α-amino acid beingoptionally substituted with one or more substituents independentlychosen from the group comprising a halogen atom and the groups NO₂, OH,CN, (C₁₋₆)alkyl, (C₂₋₆)alkenyl, (C₂₋₆)alkynyl, (C₁₋₆)alkoxy, Bzl, O-Bzland NR⁹R¹⁰, R⁹ and R¹⁰ each being independently H, O, or (C₁₋₆) alkyl;and each nitrogen atom with a peptide amide bond and the amino group ofA¹ being optionally substituted with a methyl group, it being understoodthat there is at least one such methyl group in a peptide of generalformula (III); and the pharmaceutically acceptable salts and protectedforms of such peptides.
 15. Use according to claim 13, characterized inthat the somatostatin antagonist analogue is chosen from the groupcomprising: the following peptides:Cpa-cyclo[D-Cys-Pal-D-Trp-N-Me-Lys-Thr-Cys]-D-Trp-NH₂;Cpa-cyclo[D-Cys-Tyr-D-Trp-N-Me-Lys-Thr-Cys]-Nal-N-NH₂;Cpa-cyclo[D-Cys-Pal-D-Trp-N-Me-Lys-Thr-Cys]-Nal-NH₂; the peptide knownby the code name AC-178,335; the octapeptide known by the code nameODN-8; the peptide known by the code name SB-710411; the peptide knownby the code name BIM-23056; the compound known by the code nameBN-81674; the compound known by the code name SRA-880; and theirpharmaceutically acceptable salts and protected forms.
 16. Use of asomatostatin antagonist analogue in order to support in vitro follicledevelopment.
 17. Use of a somatostatin antagonist analogue in toxicologytests relating to another compound in order to determine the presence orthe absence of an effect of slowing the follicle growth caused by saidother compound.